HNRNPA2B1-Mediated MicroRNA-92a Upregulation and Section Acts as a Promising Noninvasive Diagnostic Biomarker in Colorectal Cancer

Simple Summary Colorectal cancer (CRC) is the second leading cause of cancer deaths, with approximately 15–25% of the primary diagnosis. MicroRNA-92a (miR-92a) is considered one of the most promising biomarkers for colorectal cancer diagnosis; however, at present the diagnostic accuracy of miR-92a for CRC remains inconclusive and the upstream regulatory mechanism is not well understood in CRC development. In this study, we firstly found that serum/plasma miR-92a had better diagnostic efficacy compared to stool samples in CRC through meta-analysis. Additionally, we confirmed that decreased miR-92a expression and secretion take place in CRC cells after knockdown of heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1) in vitro. The common peaks of N6-methyladenosine (m6A) and HNRNPA2B1 in proximate miR-92a suggested HNRNPA2B1 mediated miR-92a expression through m6A modification. Thus, we identified that miR-92a derived from blood could be a better noninvasive biomarker, and provide insight into the mechanism of miR-92a in the expression and secretion in CRC. Abstract MicroRNA-92a (miR-92a) may serve as a novel promising biomarker in multiple cancers, including colorectal cancer (CRC); however, the diagnostic accuracy and the underlying molecular mechanism of miR-92a in CRC is poorly understood. We first carried out meta-analysis and found that serum/plasma miR-92a yield better diagnostic efficacy when compared to stool samples and CRC tissues, and this finding was validated by our independent study through stool sample. Multiple bioinformatics assay indicated that miR-92a expression was positively correlated with heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1) expression and closely related with the clinical characteristics of CRC. Experimental evidence showed that knockdown of HNRNPA2B1 could significantly decrease miR-92a expression and secretion in RKO cells. HNRNPA2B1 mediated miR-92a via m6A RNA modification. These findings indicate that HNRNPA2B1-m6A RNA modification-derived MicroRNA-92a upregulation and section from the local CRC acts a candidate noninvasive serum biomarker in colorectal cancer. Our study provides a novel insight into miR-92a mechanisms in relation to both expression and secretion for CRC diagnosis.


Introduction
Colorectal cancer (CRC) is the third most frequently diagnosed cancer. Aside from being the second leading cause of cancer deaths [1], diagnosis is traumatic and has lifechanging consequences. Unfortunately, 20% of those newly diagnosed have carcinoma clinical dataset based on stool samples. TP, FP, FN, and TN test results were pooled to obtain pooled sensitivity, specificity, and diagnostic odds ratio (DOR) using Stata (version 15.1). Summary receiver operator characteristic (SROC) curve and AUC were generated to provide an opportunity for visual representation.

Recruitment of Volunteers for Prospective Studies
This study was a cross-sectional blind comparison and was approved by the Ethics Committee of Cancer Hospital, Chinese Academy of Medical Sciences (ethical approval no. 22/139-3340). All subjects signed the informed consent. We prospectively enrolled 144 outpatients undergoing colonoscopy or preoperative colorectal cancer patients for opportunistic screening from April 2021 to January 2022 in Cancer Hospital of Chinese Academy of Medical Sciences. Inclusion and exclusion criteria are further displayed in Appendix B.

MiRNA Extraction and qPCR in Stool Samples
Stool sample collection and the miR-92a test were conducted with a double-blind method. Total RNA (about 0.5 g of stool) was extracted using the REColonTM Nucleic Acid Extraction kit (GeneBioHealth, Shenzhen, China) and reverse-transcribed into cDNA by adding 30 ng of RNA for each sample. The qPCR was performed with a LightCycler 480 system (Roche Diagnostics, Indianapolis, IN, USA) and a standard curve was established to calculate the copy number of each sample. Samples with a copy number lower than 902 copies/µL were interpreted as negative, and those larger than or equal to 902 copies/µL were interpreted as positive.

Bioinformatics Analysis of CRC Datasets
The gene expression profiles of total 499 CRC and 228 normal colorectum human tissue samples were retrieved from the Gene Expression Omnibus, also known as GEO, with six HNRNPA2B1 and six miRNA expression datasets (Appendix B). Exactly matched RNA-seq and miRNA expression data were obtained from TCGA datasets with a total of 545 CRC and adjacent tissues to explore the correlation between HNRNPA2B1 and miR-92a. Clinicopathological data including TNM stage, lymph node status, and metastasis status were extracted from TCGA miRNA expression datasets. The HNRNPA2B1-targeted miRNA data were selected from the research of Lee et al. [13] and 2 GEO datasets (GSE108153 and GSE49246). For the m6A and HNRNPA2B1 enrichment peaks, we retrieved data, respectively, from the GSE29714 and GSE107768 datasets.

Cell Culture In Vitro
CRC cell lines (RKO, SW480 and HCT116) were purchased from the National Infrastructure of Cell Line Resources in China (Beijing, China). MEM and 1640 medium with 10% FBS (ThermoFisher Scientific, Inc., Waltham, MA, USA) were used to incubate the CRC cell lines at 37 • C with 5% CO 2 .

m6A RNA Methylation Assay
TRIzol (ThermoFisher Scientific, Catalog number: 15596026, Waltham, MA, USA) was used to extract total RNA from SW480. The chemically fragmented RNA was incubated with m6A antibody (Synaptic Systems, 202111, Göttingen, Germany) to immunoprecipitate according to the guideline of EpiMark N6-Methyladenosine Enrichment Kit (NEB) as we previously reported [14]. Enrichment of m6A containing mRNA was then analyzed using qRT-PCR.

RNA Immunoprecipitation Assay
RIP was performed with Magna RIP RNA-binding protein immunoprecipitation kits (Millipore, 17-700, Burlington, MA, USA). Antibody against HNRNPA2B1 (Proteintech 14813-1-AP, Rosemont, IL, USA) was used. Total RNA from SW480 cell line was extracted and depleted of ribosomal RNA. The RNA protein complexes were washed and mixed with RIP immunoprecipitation buffer. Finally, RNA expression was evaluated by qRT-PCR, which was normalized to input and IgG.

SiRNA Transfection
To knockdown endogenous gene expression, two synthesized duplex RNAi oligos (Synbio Technologies) targeting human HNRNPA2B1 mRNA sequences were used, and are provided in the Appendix B (Table A3). The transfection of small interfering RNAs (siRNA) was performed using RNAiMax Reagent (Invitrogen, Waltham, MA, USA). The cells were transfected with siRNA according to the manufacturer's instructions, and the efficiency of siRNA transfection was evaluated by immunoblot analysis and qRT-PCR.

Secreting miRNA Isolation
Five mL RKO cell culture medium was collected from MEM medium with 10% exosome depleted FBS (VivaCell, Yerevan, Armenia) which was changed one day before the transfection with siRNA or siNC treatment. Then, siRNAs were transiently transfected into RKO cells for 0, 24, and 48 h, and total RNA from medium was extracted using exoEasy maxi Kit (QIAGEN, Germantown, MD, USA) to collect the secreting miRNA according to the manufacturer's protocol.

RNA Extraction, Reverse Transcription, and RT-qPCR
After extracting the RNA from adherent cells, exfoliated cells and culture medium were collected for RNA extraction by TRIZOL regent as we previously reported [15], and cDNAs were synthesized with PrimeScript™ IV 1st strand cDNA Synthesis Mix (Takara, Beijing, China) for mRNA and Mir-X™ miRNA First-Strand Synthesis (Takara Bio USA, Inc., San Jose, CA, USA) for miRNA. QRT-PCR analysis was measured using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China). Each assay was carried out in triplicate by the Light Cycler 480 Instrument (Roche, Basel, Switzerland). The primers for RT-qPCR are provided in the Appendix B (Table A4).

Statistical Analysis
All statistical analyses were performed by GraphPad Prism 9 (GraphPad Software, Inc.). Continuous variables were presented as means with SDs and the unpaired two-tailed Student's t-test or Wilcoxon's tests, as well as Spearman's method, were used to compare differences between two groups with a significance of p < 0.05.

MiR-92a Was Differentially Expressed among Tissue, Blood, and Stool Sample Types in CRC
Initially, we systematically searched and reviewed studies related to miR-92a in CRC. A total of 204 studies were retrieved from public databases and 78 duplicates were immediately excluded. After screening titles and abstracts, 66 articles were considered eligible for data extraction. After reviewing the articles in detail, 37 further studies were excluded due to lack of diagnostic data or irrelevant researches. A total of 30 studies containing only stool-based data were eventually deemed pertinent [7,8,. The flowchart presented below provides the process of inclusion/exclusion for this systematic review and meta-analysis ( Figure 1). The total included studies involved 2345 CRC patients and 1954 controls. The types of specimens were classified as tissue (n = 16), plasma or serum (n = 13), and stool (n = 6), shown in Table 1. The weighted fold changes (FC) were 2.64 ± 1.30, 5.07 ± 3.07, and 28.42 ± 49.09, separately, for tissue, stool, and blood (including plasma and serum). This confirmed the feasibility of miR-92a as an optional diagnostic biomarker, which was highly expressed in CRC with three sampling methods, especially with blood and stool sampling.

Diagnostic Accuracy of miR-92a Based on Plasma/Serum Appears Better than Stool Samples
Previous studies suggested that miR-92a was highly expressed in CRC, indicating a potential effective diagnostic marker. Compared with colonoscopy and biopsy, blood or stool examination is a more convenient and effective choice for patients. However, which is better for diagnosis efficiency is still confusing. In order to explore this issue, we included a total of 19 studies (blood = 13, stool = 7) for further meta-analysis. There were 835 CRC patients and 598 controls in the plasma/serum group, and 838 CRC patients and 793 controls in the stool sample group (Table 2). It is worth mentioning that one included article provided both plasma-and stool-based miRNA data [8].

MiR-92a as a Potential Biomarker in the Independent Prospective Data from Stool Samples
To further verify the clinical efficacy of miR-92a as a diagnostic biomarker, we recruited a total of 144 volunteers for opportunistic screening (CRC = 72, healthy individuals = 72), including 82 males and 62 females. The demographic and clinicopathological characteristics of the enrolled CRC patients and healthy volunteers are displayed in Appendix A, Tables A1 and A2. Compared to the normal group, the miR-92a expression was significantly increased in colorectal cancer group with 1.36-fold change (Figure 3a). The diagnostic efficiency of miR-92a test in CRC patients was evaluated through ROC analysis, and the AUC was 0.861 (95% CI: 0.801-0.901, p < 0.05) with a sensitivity/specificity of 0.889/0.736, suggesting that this noninvasive detection method had a desired diagnostic ability for colorectal cancer (Figure 3b). Next, as shown in the histograms, the positive detection rate of miR-92a also increased with lymph node metastasis (Figure 3c), tumor stage development (Figure 3d), and tumor size increasing (Figure 3e). In summary, with the progression and metastasis of colorectal cancer, the differential expression of miR-92a becomes higher, indicating that miR-92a could be a promising biomarker for CRC.

HNRNPA2B1 May Regulate miR-92a through m6A Modification in Colorectal Cancer
Further, we would like to investigate the reason for the elevated miR92a based on blood and stool samples in CRC, so as to better understand the mechanisms. Recently, Alarcón et al. [11] reported that HNRNPA2B1 could affect the production of miRNAs by mediating primary microRNA processing. Thus, we hypothesized that HNRNPA2B1 might be involved in the progression of CRC by regulating miR-92a expression. To explore the mechanism of m6A regulator HNRNPA2B1 and miR-92a in CRC, we first analyzed the expression profiling of m6A reader HNRNPA2B1 in six GEO datasets with 193 CRC and 100 normal colorectal tissues, showing that HNRNPA2B1 was highly expressed in CRC patients, and a similar result was displayed in miR-92a expression from six other GEO datasets (CRC = 306, the normal = 128), shown in Figure 4a,b. Next, strong positive correlations between HNRNPA2B1 and miR-92a were identified in the TCGA dataset (R = 0.352, p < 0.001, Figure 4c). Similar to the results of the validated stool samples, miR-92a expression from the TCGA database also had the higher expression with tumor stage development (Figure 4d), lymph node metastasis (Figure 4e), and distant metastasis (Figure 4f).
Notably, 12 miRNAs overlapped by increasing miRNAs in CRC (GSE108153 and GSE49246) and HNRNPA2B1-targeted miRNAs (Lee's study), which included miR-92a (Figure 4g). The enrichment peak of m6A and HNRNPA2B1 from GSE29714 and GSE107768 demonstrated the colocalization of HNRNPA2B1 and m6A modification nearby miR-92a sites (Figure 4h). To address whether HNRNPA2B1 regulates miR-92a expression through m6A modification, in vitro experiments were employed via m6A RNA methylation tests and RNA immunoprecipitation tests. Through RIP-qPCR analysis, the Figure 4i  (d-f) the relationship between miR-92a expression and clinical characteristics of TNM stage, lymph node metastasis, and distant metastasis, respectively; (g) the Venn diagram of HNRNPA2B1-targeted miR-92a from the GEO datasets (GSE108153, GSE49246) and Lee's study; (h) the enrichment peak of m6A and HNRNPA2B1 in miR-92a sites from the HITS CLIP sequence; (i) RIP-qPCR showed that the m6A and HNRNPA2B1 peak region was occupied by the nearby miR-92a sites in SW480 cells. * p < 0.05; *** p < 0.001; **** p < 0.0001.

HNRNPA2B1 May Regulate the Expression and Secretion of miR-92a In Vitro
To the best of our knowledge, miR-92a was produced by the sites of tumor tissue, and then exfoliated into the intestinal tract or secreted into the blood circulation system. Our previous finding demonstrated that the expression of miR-92a in CRC from tissue, stool, and blood was extremely high and miR-92a co-immunoprecipitate was regulated by HNRNPA2B1. In this way, we next wondered whether HNRNPA2B1 could mediate the expression and secretion of miR-92a in CRC, through in vitro experiments. As Figure 5a displays, the model diagram mimicked the process of miR-92a shedding from tumor tissue (adherent cells) into the intestine by detecting MiR-92a expression in stool samples (exfoliated cells), and the process of entering the bloodstream through the circulatory system (secreting RNA). Firstly, we established transient HNRNPA2B1 knockdown models in RKO cells with the siRNA sequence. Figure 5b,c evidences the successful knockdown of HNRNPA2B1, respectively, in protein and mRNA levels in RKO cells. Similar results in HCT116 and SW480 cell lines are shown in Figure A1. Then, we compared the expression of miR-92a in RKO adherent and exfoliated cells. The results confirmed that miR-92a was significantly downregulated in CRC with the knockdown of HNRNPA2B1 in protein and RNA level (Figure 5d,e). Finally, after the treatment of RKO cells at different time periods (0 h, 24 h, and 48 h), it was found that the secretion of miR-92a was significantly reduced after knockdown of HNRNPA2B1 (Figure 5f), indicating that HNRNPA2B1 may regulate the secretion of miR-92a by controlling the expression of miR-92a.

Discussion
Numerous primary and secondary studies have found that early detection and diagnosis will decrease CRC-specific mortality compared with no screening [43]. However, due to invasiveness, associated costs, and stigma, colonoscopy is often refused by patients, and postponing this investigation can have a devastating impact on outcomes once diagnosed. The public may be more willing to endure less invasive sampling methods such as blood and stool collections. Therefore, we need to ensure we are using the most effective, least invasive technique for tumor screening. As routine screening methods, guaiac-based fecal occult blood tests (gFOBT), fecal immunochemical test (FIT), and serum-based SEPT9 DNA methylation test (Epi proColon) have relatively low sensitivities for CRC [44]. Additionally, the current multitarget stool DNA test is not considered cost-efficient, and there is a lack of evidence from large-scale prospective studies. Furthermore, colorectal cancer is a highly heterogeneous clinical entity; genetic KRAS, NRAS, BRAF, or HER2 genes also work as the critical diagnostic and prognostic biomarkers. MiRNAs regulate critical pathways involved in the CRC pathogenesis, including the p53, PI3K, RAS, MAPK, and EMT transcription factors, and Wnt/β-catenin pathways. MiRNAs could be a further therapeutic potential to explore effective targeting of KRAS-mutant CRC [45]. Therefore, it is important to consider diagnostic biomarkers, especially those associated with tumorigenesis, such as microRNA, as they may provide insight into multiplicity and proliferation.
Lin et al. summarized human genome regulation and carcinogenesis diversity which are closely associated with miRNAs from tissue sample source [46]. For plasma/serum samples, through the systemic circulatory system, miRNAs are secreted or shed from the local tumor tissue and transported to the blood packed in exosomes or combined with biomacromolecules [47,48]. Regarding stool samples, miRNAs are directly derived from exfoliated colonocytes through the intestinal cavity [49]. MiRNAs extracted from stool samples are easy to obtain from patients, and have been demonstrated to have high quality [50]. Therefore, several researchers have suggested that miRNAs based on blood or stool have potential as diagnostic biomarkers, specifically for CRC. However, even though several studies have reported the efficacy of miR-92a as a CRC diagnostic biomarker, no one has focused on systematically analyzing miR-92a derived from blood or stool samples in order to determine which one can better assess the risk of colorectal cancer.
We firstly synthesized evidence and compared the miR-92a diagnostic value for colorectal cancer based on plasma/serum and stool samples. We found that the diagnostic efficiency of miR-92a based on blood samples appears better compared to stool samples. Interestingly, there was no diagnostic efficiency difference of miR-92a expression level between plasma and serum samples through meta-regression analysis (data not shown). We were also able to validate that miR-92a could be used to distinguish colorectal cancer patients from controls, yielding an AUC of 0.861 with pooled sensitivity of 89%. However, pooled specificity dropped to around 74% in the prospective element of this study when independently verifying with external data, whereas under meta-analysis, we found that the stool sample group had an AUC of 0.847 with an 84% specificity. However, this time, sensitivity dropped to 68%, which needs to be considered in more detail. For example, the stool itself may be more heterogeneous and more difficult to standardize across a small sample of individuals. As described previously, sampling different parts of the stool itself is also likely to influence the within-specimen variations [51]. Additionally, for stool samples, it is not always possible to have exact exposure times when exfoliated cancer cells fall from the colorectum. This may result in a number of surviving cells being reduced in the stool. In addition, compared with phlebotomy, which is conducted in clinics, stool specimens tend to be prepared at home and unaided, which adds to the number of potentially confounding factors.
Since blood-and stool-derived miR-92a has been identified as a promising biomarker for CRC, we further investigated the upstream regulatory mechanisms of miR-92a. This was performed to better understand the underlying mechanisms involved in miR-92a elevation. Previous research has shown that HNRNPA2B1 could interact with microRNA and its complex protein DGCR8 to promote the miRNA processing through binding with the m6A mark [11]. In addition to this, Villarroya et al. found that HNRNPA2B1 can control miRNA sorting through binding to the sumoylated special motifs [52]. It is worth mentioning that O'Grady et al. considered that the regulation of RNA profile in the cell was generally thought of as a balance between transcription and decay. They found that HNRNPA2B1 might regulate the export the secreting miRNA to control the intracellular abundance [53]. The regulatory mechanisms of miR-92a on HNRNPA2B1 are varied, and it is therefore important that we focus on gaining insights into these mechanisms of action.
As one of the most important epigenetic modifiers in eukaryotes, m6A modification may affect miRNA splicing and carcinogenic maturation [54]. Researchers have also previously found that loss of methyltransferase, such as METTL3, causes downregulation of m6A modification levels for miR-100 and miR-125b. This process thereby inhibits miRNA expression and possibly causes specific drug resistances [55]. Yue et al. found that miR-96 promotes the initiation and progression of colorectal cancer by modulating the AMPKα2-FTO-m6A/MYC axis, which again highlights the underlying mechanisms involved in CRC development [56]. Regarding the m6A reader, IGF2BP2 interfered with RAF-1 degradation via miR-195 and promoted proliferation and survival of CRC cells [57]. Additionally, miR-6125 knockdown is thought to promote CRC proliferation through YTHDF2-dependent recognition of m6A-modified GSK3β [58]. Taken together, these findings provide evidence that there is an interaction between m6A modification and miRNA in CRC development. Combined with the mechanism of HNRNPA2B1 in the regulation of miRNA secretion, we speculate that HNRNPA2B1 may regulate the expression of miR-92a through m6a modification in colorectal cancer.
In vitro experiments illustrate that the expression level of miR-92a significantly decreased after knockdown of HNRNPA2B1. In addition, we closely monitored miR-92a in adherent cells, exfoliated cells, and those secreted from cell culture mediums for the expression level of miR-92a based on tissues, stool, and blood sample types, and we found that HNRNPA2B1 did regulate the expression and secretion of miR-92a. Further, RIP-qPCR experiments confirmed the existence of HNRNPA2B1 and m6A-modified sites adjacent to miR-92a. These results infer that HNRNPA2B1 may be enriched near miR-92a sites through m6A recognition, so as to regulate the expression and secretion of miR-92a. Taken together, these results contribute to our understanding of heterogeneous diagnostic miR-92a-based efficacy. This research also garners insight into the regulatory mechanisms involved in HNRNPA2B1 and m6A on miR-92a in CRC.

Conclusions
In summary, we initially conducted a diagnostic meta-analysis to compare plasma/serumbased miR-92a with stool-based samples, drawing the conclusion that miR-92a from blood sources has superior diagnostic efficacy at distinguishing CRC patients from healthy controls. The expression and secretion of miR-92a appears to be regulated by HNRNPA2B1 through m6A modification, which ultimately helps to individualize colorectal cancer diagnosis and care. Figure A1. Knockdown experiments confirmed that HNRNPA2B1 may regulate the expression of miR-92a. (A,B) HNRNPA2B1 expression was successfully knocked down by si-HNRNPA2B1 transfection for 48 h in HCT116 and SW480 cells; (C,D) HNRNPA2B1 expression in HCT116 and SW480 cells with or without HNRNPA2B1 knockdown was measured by qRT-PCR (n = 3); (E,F) miR-92a expression in HCT116 and SW480 cells with or without HNRNPA2B1 knockdown (n = 3), respectively. * p < 0.05; *** p < 0.001; **** p < 0.0001.